Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Paraneoplastic pemphigus associated with fatal bronchiolitis obliterans and intractable mucosal erosions: Treatment with cyclosporin in addition to steroid, rituximab and intravenous immunoglobulin.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that presents as severe mucosal erosions and variable cutaneous lesions and is primarily associated with hematologically malignant or benign diseases. A 59-year-old Japanese woman presented with oral, ocular and vaginal mucosal erosions and erythema as well as blistering on her trunk and limbs. She developed bronchiolitis obliterans; lymphadenopathy in the cervical, subclavian, para-aortic and intraperitoneal regions; and splenomegaly. PNP with B-cell lymphoma was diagnosed. She was treated with two courses of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) for B-cell lymphoma, rituximab once every 3 weeks for five cycles, steroid pulse therapy, oral prednisolone, cyclosporin and high-dose i.v. immunoglobulin. The B-cell lymphoma was in remission after two courses of R-CHOP treatment. Although her skin erythema and blistering were also improved, the mucosal erosions and bronchiolitis obliterans gradually worsened. The patient died of bronchiolitis obliterans after 6 months of hospitalization. Because a cellular immune response is thought to be involved in the pathogenesis of PNP, cyclosporin therapy is expected to aid in suppressing the cellular response. In this case, however, the patient's mucosal lesions and bronchiolitis obliterans were not improved by regular administration of cyclosporin therapy.

Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

"Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1ß in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1ß at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

Infantile generalized pustular psoriasis: successful disease control with intermittent etretinate.

Infantile generalized pustular psoriasis is a rare form of psoriasis and the best treatment is controversial. We experienced a 2-year-old female with erythema on her neck and axilla starting at 3 months of age. She presented with recurrent annular and geographic scaly erythema with a few pustules on the neck, precordium and axilla, but no fever. The histopathology revealed subcorneal neutrophilic infiltration and microabscesses without Kogoj's spongiform pustules. The initial diagnosis was subcorneal pustular dermatosis. However, she developed widespread geographic erythema and numerous pustules over her entire body with a fever when she got a cold. A second skin biopsy revealed monolocular pustules and Kogoj's spongiform pustules in the subcorneal layer. Etretinate was administrated after a diagnosis of pustular psoriasis was made and her condition improved gradually. The choice of treatment depends on patient age, general condition and the disease severity.

CCL27 is downregulated by interferon gamma via epidermal growth factor receptor in normal human epidermal keratinocytes.

The cutaneous T cell-attracting chemokine (CTACK)/CCL27 is indispensable in skin inflammation. CTACK/CCL27 is exclusively produced by epidermal keratinocytes to attract CCR10-expressing T lymphocytes to the skin. We investigated the mechanism of CTACK/CCL27 production from normal human epidermal keratinocytes (NHEKs) by the proinflammatory cytokines TNFα and IFNγ. CTACK/CCL27 production was induced by TNFα via ERK, JNK, p38, and NFκB. The induction of CTACK/CCL27 by TNFα was suppressed by IFNγ via a pathway dependent on JAK, STAT1, and STAT3. Our results also demonstrated that IFNγ and TNFα induced the phosphorylation of EGFR and the following phosphorylation of ERK, which is partly responsible for the suppressive effect of IFNγ on TNFα-induced production of CTACK/CCL27. Peri-lesional skin of psoriasis demonstrates early inflammatory changes as we have previously reported. CTACK/CCL27 expression was diffuse in the peri-lesional epidermis, while it was restricted to basal layer in lesional epidermis, suggesting that CTACK/CCL27 expression was induced in the early stage of psoriatic plaque formation, and IFNγ could participate in the suppression of CTACK/CCL27 expression in the lesional epidermis, reflecting the later stage of psoriatic plaque formation. Our study suggests that CTACK/CCL27 may have a pivotal role in the early stage of psoriasis plaque formation, but should be downregulated in the later stage to induce inflammation characteristic for chronic psoriasis plaques.

Eccrine sweat contains IL-1α, IL-1ß and IL-31 and activates epidermal keratinocytes as a danger signal.

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1ß, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1ß by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents.

BACKGROUND: During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. OBJECTIVE: In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. METHODS: We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-ß (TGF-ß) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. RESULTS: The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-ß1 expression in the epidermis. The inhibition of TGF-ß kinase completely suppressed α-SMA expression in the dermal matrix. CONCLUSIONS: Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-ß1, which is responsible for myofibroblast differentiation.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

Mite allergen is a danger signal for the skin via activation of inflammasome in keratinocytes.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disorder caused by multiple factors. Among them, house dust mite (HDM) allergens are important in the development of AD. In airway allergy, HDM allergens activate innate immunity. However, information regarding the activation of innate immunity by HDM allergens in the skin is limited. OBJECTIVES: The inflammasome is a key regulator of pathogen recognition and inflammation. We investigated whether HDM allergens activate the inflammasome in epidermal keratinocytes. METHODS: Keratinocytes were stimulated with Dermatophagoides pteronyssinus, and the activation of caspase-1 and secretion of IL-1ß and IL-18 were examined. Formation of the inflammasome was studied by analyzing the subcellular distributions of inflammasome proteins. The importance of specific inflammasome proteins was studied by knocking down their expression through transfection of keratinocytes with lentiviral particles carrying short hairpin RNAs (shRNAs). RESULTS: D pteronyssinus activated caspase-1 and induced caspase-1-dependent release of IL-1ß and IL-18 from keratinocytes. Moreover, D pteronyssinus stimulated assembly of the inflammasome by recruiting apoptosis-associated specklike protein containing a caspase-recruitment domain (ASC), caspase-1, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin-domain containing 3 (NLRP3) to the perinuclear region. Finally, infection with lentiviral particles carrying ASC, caspase-1, or NLRP3 shRNAs suppressed the release of IL-1ß and IL-18 from the keratinocytes. Activation of the NLRP3 inflammasome by D pteronyssinus was dependent on cysteine protease activity. CONCLUSION: House dust mite allergens are danger signals for the skin. In addition, HDM-induced activation of the NLRP3 inflammasome may play a pivotal role in the pathogenesis of AD.

Desmosome disassembly in response to pemphigus vulgaris IgG occurs in distinct phases and can be reversed by expression of exogenous Dsg3.

Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupts adhesion is not fully understood. To address this issue, primary human keratinocytes (KCs) and patient IgG were used to define the morphological, biochemical, and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living KCs demonstrated that PV IgG cause rapid Dsg3 internalization, which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde manner along with these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent-insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.

PPARγ mediates innate immunity by regulating the 1α,25-dihydroxyvitamin D3 induced hBD-3 and cathelicidin in human keratinocytes.

BACKGROUND: Production of antimicrobial peptides (AMPs) is the primary mechanism by which skin innate immunity protects against infection. Hormonally active vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D3) is a vital regulator of skin innate immunity, and has been shown to increase the expression and function of AMPs. OBJECTIVE: PPARγ is a ligand-activated nuclear receptor and plays a role in keratinocyte differentiation and cutaneous homeostasis. In this study, we investigate whether 1,25D3-activated PPARγ signaling regulates AMP expression in keratinocytes. METHODS: Subconfluent keratinocytes were treated with 1,25D3 for the indicated times. The mRNA and protein levels of AMPs were detected by RT-PCR and Western blot, and the DNA binding activation of PPARγ, VDRE and AP-1 was investigated by EMSA. To examine the role of PPARγ, the recombinant adenovirus carrying a dominant-negative form of PPARγ (dn-PPARγ) was constructed and transfected into keratinocytes. RESULTS: We show here that 1,25D3 significantly enhances hBD-3 and cathelicidin expression in keratinocytes. Expression of dn-PPARγ did not affect binding to the vitamin D-responsive element (VDRE), which is crucial for cathelicidin induction by VD3; however, it did decrease 1,25D3 induction of both hBD-3 and cathelicidin. Inhibition of the p38, ERK, and JNK signaling pathways blocked hBD-3 expression, whereas only p38 inhibition suppressed cathelicidin induction. dn-PPARγ had no effect on ERK and JNK activity, but inhibited p38 phosphorylation and suppressed 1,25D3-induced AP-1 activation via effects on Fra1 and c-Fos proteins. CONCLUSIONS: In conclusion, PPARγ regulates the 1,25D3-induced hBD-3 and cathelicidin expression in keratinocytes through the regulation of AP-1 and p38 activity.

E2 Polyubiquitin-conjugating enzyme Ubc13 in keratinocytes is essential for epidermal integrity.

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth, the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.

Inflammatory mediator TAK1 regulates hair follicle morphogenesis and anagen induction shown by using keratinocyte-specific TAK1-deficient mice.

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the NF-kappaB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial-mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7(fl/fl)K5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7(fl/fl)K5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7(fl/fl)K14-Cre-ER(T2)). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.

Living skin equivalents constructed using human amnions as a matrix.

BACKGROUND: Living skin equivalents (LSEs) are being used to treat burn wounds, skin defects, and chronic wounds, and today, several biomaterials are applied as dermal matrices in LSEs. The amnionic membrane (AM) is known to have useful properties as a dermal matrix and can be used to construct a LSE. OBJECTIVE: To develop a new LSE with human AM as the matrix. METHODS: Human AM was de-epithelialized and investigated to determine whether it supported keratinocyte adherence and proliferation, and fibroblast in-growth and proliferation. A new LSE was constructed by seeding keratinocytes on the epithelial side of fibroblast-populated, de-epithelialized AM and was investigated histologically. The LSE was transplanted onto a full-thickness wound on a nude mouse and a histological examination was conducted. RESULTS: De-epithelialized AM supported the adherence and proliferation of keratinocytes and the in-growth of fibroblasts. The new LSE demonstrated good mechanical properties and revealed good morphology, with a well differentiated epidermis and well developed basement membrane. The LSE grafts survived well on nude mice, showing good morphology. CONCLUSION: A LSE with amnions as a matrix exhibited good morphology, low cost, and good mechanical properties and may be useful as a skin substitute for clinical use.

Nodal lymphangiogenesis and metastasis: Role of tumor-induced lymphatic vessel activation in extramammary Paget's disease.

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression.

IL-20 cytokine subfamily members, including IL-19, IL-20, and IL-24, are highly expressed in psoriatic skin lesions. Here, we demonstrate that psoriasis mediators IL-17 and IL-22 synergistically induce the production of IL-20 subfamily proteins in cultured human keratinocytes. Interestingly, expression of the IL-22 receptor (IL-22R) also increased in epidermal lesions versus normal skin. IL-22R over-expression using an adenoviral vector to mimic psoriatic conditions in cultured keratinocytes significantly enhanced IL-17- and IL-22-induced production of IL-20 subfamily cytokines. Furthermore, IL-17 and IL-22 coordinately enhanced MIP-3alpha, IL-8, and heparin-binding EGF-like growth factor (HB-EGF) production, depending on the amount of IL-22R expression. Additionally, because IL-20 and IL-24 share the IL-22R with IL-22, the function of IL-20 and IL-24 was also increased. IL-20 and IL-24 have effects similar to that of IL-22; IL-24 showed more potent expression than IL-20. A combination of IL-24 and IL-17 increased the production of MIP-3alpha, IL-8, and HB-EGF, as did a combination of IL-22 and IL-17. These data indicate that increased IL-22R expression in epidermal keratinocytes contributes to the pathogenesis of psoriasis through enhancing the coordinated effects of IL-22 and IL-17, inducing the production of the IL-20 subfamily, chemokines, and growth factors.

Ramified microglial cells promote astrogliogenesis and maintenance of neural stem cells through activation of Stat3 function.

The differentiation and proliferation of neural stem cells (NSCs) are regulated by a combination of their intrinsic properties (e.g., transcription factors, epigenetic factors, and microRNA regulation) and cell-extrinsic properties from the microenvironment around NSC (e.g., cytokines, growth factors, and cell-cell contact). Recently, there has been a great interest in clarifying the mechanism of the influence of the microenvironment on NSCs, especially cell-cell contact between NSCs and other types of cells nearby. In this study, we investigated whether microglial (Mi) cells influence the fate of NSCs. Coculture study showed that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs through their paracrine effects. This microglia-induced astrogliogenesis was inhibited by AG490 and by overexpression of the dominant-negative form of Stat3 and SOCS3. Promoter assay revealed transactivation of Stat3 function in NSCs by Mi cells. Gene expression study revealed that mRNA of Notch family members (notch1-3) and sox9 in NSCs was significantly upregulated by Mi cells, and this up-regulation was inhibited by AG490. These results demonstrated that ramified Mi cells promoted astrogliogenesis and maintenance of NSCs by activating Stat3 function and via notch and sox9 signaling pathways.